1. Field of the Invention
The present invention relates to a scanning laser microscope which condenses laser beam by using an object lens, irradiates a sample with spot light of the condensed laser beam, spectral-decomposes light such as fluorescence from the sample, and detects fluorescence in an arbitrary band area from the obtained spectrum or acquire spectral data.
2. Description of the Related Art
A scanning laser microscope can excite a fluorescent reagent by irradiating a sample dyed to the fluorescent reagent with laser beam, spectral-decomposes fluorescence emitted from the sample, and detect the fluorescent in an arbitrary band area from the obtained spectrum or acquire spectral data of the fluorescence from the spectrum. Such a scanning laser microscope is disclosed in, e.g., U.S. publication No. 6614526 and Jpn. Pat. Appln. KOKAI Publication No. 2000-56244.
U.S. Publication No. 6614526 discloses the following technique. Fluorescence from a sample is dispersed by using a prism, the dispersed fluorescence is condensed by using a focusing optical system so as to enter a slit/detector arrangement. The slit/detector arrangement has a reflection surface forming a slit aperture diaphragm, divides the fluorescence condensed by the focusing optical system into a plurality of partial beam corresponding to respective spectral areas by using the reflection surface, and forms images of the respective partial beam in respective detectors. The slit/detector arrangement can vary a band area to be assigned to each detector by driving and controlling the reflection surface forming the slit aperture diaphragm.
Jpn. Pat. Appln. KOKAI Publication No. 2000-56244 discloses the following technique. Fluorescence from a sample is image-formed in a spectrum form by using a combination of a diffraction grating and a concave mirror or a combination of a prism and a condenser lens. An array of light-deflecting micro mirrors (which will be referred to as a DMD hereinafter) is arranged at this image formation position.
The DMD is composed of a plurality of micro mirrors in an array form. The DMD has a structure capable of electrically switching an angle of a reflection surface of each micro mirror, i.e., switching a reflection direction between two directions. The DMD selects a band area of image-formed fluorescence in a spectrum form. The fluorescence in the selected band area enters a photodetector. In Jpn. Pat. Appln. KOKAI Publication No. 2000-56244, a photodetector is arranged to at least one of two reflection directions of the DMD, and light in an arbitrary band area are led to the photodetector from a spectrum image-formed on the micro mirror. Further, in Jpn. Pat. Appln. KOKAI Publication No. 2000-56244, spectral characteristics of fluorescence can be detected by sequentially switching the micro mirrors.
The scanning laser microscope uses a beam splitter in order to separate laser beam as exciting light and fluorescence from a sample. In the scanning laser microscope, a plurality of types of beam splitters are prepared in accordance with wavelength characteristics of laser wavelengths and fluorescence used for observation. Any one of the plurality of beam splitters is arranged on a light path in accordance with observation of a sample.
For example, when a fluorescent reagent of a sample is excited with Ar laser beam having a wavelength of 488 nm in order to obtain fluorescence having a wavelength of 500 nm to 600 nm, there is used a dichroic beam splitter having characteristics which reflect light having a wavelength of 488 nm and transmit a band area with a wavelength of 500 nm to 600 nm therethrough.
When a fluorescent reagent of a sample is excited with Ar laser beam having a wavelength of 515 nm in order to obtain fluorescence having a wavelength of 530 nm to 650 nm, there is used a dichroic beam splitter having characteristics which reflect light having a wavelength of 515 nm and transmit a band area with a wavelength of 530 nm to 650 nm therethrough.
The plurality of beam splitters are provided to a turret formed into, e.g., a discoid shape. The turret rotates around a rotary bearing portion. The turret arranges any one beam splitter on an optical axis by a switching operation based on rotation.
The turret has a manufacture error in swing of the rotary bearing portion, flatness of a surface on which the plurality of beam splitters are provided and others. Therefore, when the beam splitters are switched by rotating the turret, an angle of a reflection surface relative to the optical axis of each beam splitter differs depending on each beam splitter. It is to be noted that such an angle is referred to as an angular difference between the respective beam splitters.
Since there is an angular difference between the respective beam splitters, when the beam splitters are switched, light from a sample are transmitted through the beam splitter, and an angle of light which enter a dispersive element such as a prism varies. As a result, a spectrum image position obtained by dispersion by the dispersive element and condensation by a condenser lens is displaced in a spectrum direction.
A displacement of the spectrum image formation position will now be described with reference to FIG. 6. A prism 1 disperses incident light 2 of parallel light. The dispersed incident light 2 are condensed by a condenser lens 3. As a result a spectrum is image-formed on an image formation surface 4 formed by the condenser lens 3.
A variable slit 5 is arranged on the image formation surface 4. The variable slit 5 can vary a slit width in the same direction as the spectrum direction. The variable slit 5 can move a slit central position in the same direction as the spectrum direction.
A wavelength width to be taken into a photodetector 6 is changed by varying a width of the variable slit 5. A wavelength center of a band area to be taken into the photodetector 6 is changed by moving a slit center of the variable slit 5. Only light in a band area which has been transmitted through the variable slit 5 are detected by the photodetector 6.
As to the prism 1, a glass material thereof is, e.g., PBH8 and a shape thereof is an equilateral triangle. The incident light 2 enter the prism 1 through a beam splitter. The incident light 2a has a wavelength of, e.g., 510 nm (refractive index: 1.730774), and enter the prism 1 at an angle of 60° relative to a normal line a1 of an incident surface of the prism 1. An outgoing radiation angle of outgoing light 7a emitted from the prism 1 is 59.85° relative to a normal line a2 of an outgoing radiation surface.
When the beam splitter is switched to another beam splitter, the incident light 2b which enter the prism 1 through the beam splitter shifts at angle of, e.g., 6′ relative to an optical axis of the incident light 2a. Therefore, an outgoing radiation angle of outgoing light 7b emitted from the prism 1 is 59.75° relative to the normal line a2 of the outgoing radiation surface.
The spectrum image formation position depends on only an angle of outgoing light if the light which enter the condenser lens 3 are parallel light. On the image formation surface 4, a first spectrum image formation position obtained when the incident light 2a having a wavelength of 510 nm enter is different from a second spectrum image position obtained when the incident light 2b having the same wavelength but a different incident angle enter.
For example, assuming that a focal distance of the condenser lens 3 is 30 mm, a displacement quantity ΔL between the first and second spectrum image positions can be represented by the following expression.
                                                                        Δ                ⁢                                                                  ⁢                L                            =                                                (                                                            tan                      ⁢                                                                                          ⁢                      59.85                      ⁢                      °                                        -                                          tan                      ⁢                                                                                          ⁢                      59.75                      ⁢                      °                                                        )                                ×                30                                                                                        =                              0.207                ⁢                                                                  ⁢                mm                                                                        (        1        )            
On the contrary, a wavelength of light outgoing at an angle of the outgoing light 7a (outgoing at 59.85° relative to the normal line a2 of the outgoing radiation surface) before switching the beam splitter with the light which have entered at an angle of the incident light 2b is 505 nm (refractive index: 1.731626).
Therefore, when the beam splitter is switched to another beam splitter in a state that a slit central position of the variable slit 5 is set to a wavelength of 510 nm, a wavelength at the slit central position of the variable slit 5 is 505 nm. As a result, the wavelength of the light which are transmitted through the variable slit 5 and detected by the photodetector 6 is shifted by 5 nm from 510 nm to 505 nm.
In order to spectral-decompose fluorescence emitted from a sample and obtain spectral data of fluorescence from a spectrum, a width of a band area to be taken out is reduced by setting a slit width of the variable slit 5 to be narrow, and a change in light intensity in a spectrum direction is detected while moving the variable slit 5 in the spectrum direction. Therefore, even a small displacement of the spectrum image position affects an accuracy of spectral data. A displacement of the spectrum image position is a serious problem when obtaining accurate spectral data.